Determining the Role of Vitronectin in Bacterial Resistance to Bactericidal Activity of Human Serum

All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines.

1. Analysis of Vn-dependent Resistance to the Bactericidal Activity of Human Serum

1. Purchase normal human serum (NHS) from a commercial source. Prepare vitronectin or Vn-depleted serum (VDS). Replenish the VDS with 180 nM Vn which is equivalent to Vn present in NHS.
2. Prepare heat-inactivated serum (HIS) by heating NHS at 56 °C for 30 minutes in order to inactivate complement proteins.    
   NOTE: The optimal serum concentration (5%) and incubation time (15 min) for the assay were determined empirically for Haemophilus influenzae type f (Hif); these parameters could vary for other bacterial pathogens.
3. Culture bacteria (in this case, Hif M10 and the mutant Hif M10Δlph) to mid-log phase (OD₆₀₀ = 0.3). Pellet bacteria by centrifugation at 3,500 x g for 10 min.
4. Resuspend the bacterial pellet with 1 volume of dextrose gelatin Veronal buffer (DGVB⁺⁺; pH 7.3) containing 2.5% (w/v) glucose, 2 mM magnesium chloride, MgCl₂, 0.15 mM calcium chloride, CaCl₂, and 0.1% (wt/vol) gelatin.
5. Add 1.5 x 10³ colony forming unit (CFU) of bacteria to 100 µL of DGVB⁺⁺ containing 5% serum (NHS, HIS, VDS, or VDS+180 nM Vn). Incubate the sample at 37 °C for 15 min with shaking at 300 rpm.
6. Remove a 10 µL aliquot from the reaction mixture at 0 min (T₀ sample) and 15 min (T_t sample) and plate on chocolate agar. Incubate the plate at 37 °C overnight.
7. After incubation, count the colonies appearing on the plate. Calculate the percentage of bacteria killed using the following equation: (colony forming unit or CFU at T_t)/(CFU at T₀) × 100.